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系統識別號 U0002-0507201113271300
中文論文名稱 (I) 啤酒酵母菌YNR029Cp的表現和純化及甲基化活性之研究 (II) 過度表現啤酒酵母菌ADH3p對其粒線體形態影響
英文論文名稱 (I) Study of Saccharomyces cerevisiae YNR029Cp:experssion, purification, and methylation activity. (II) Effect of overpressing Saccharomyces cerevisiae ADH3p on mitochondrial morphology.
校院名稱 淡江大學
系所名稱(中) 化學學系碩士班
系所名稱(英) Department of Chemistry
學年度 99
學期 2
出版年 100
研究生中文姓名 陳宏仁
研究生英文姓名 Hang-Jen Chern
學號 698180063
學位類別 碩士
語文別 中文
口試日期 2011-06-17
論文頁數 69頁
口試委員 指導教授-陳銘凱
中文關鍵字 甲基轉移酶  甲基化  粒線體形態  酒精去氫酶III 
英文關鍵字 Methyltransferase  methylation  mitochondrial morphology  ADH3 
學科別分類 學科別自然科學化學
中文摘要 表觀遺傳調控(epigenetic regulation),泛指在沒有改變DNA序列的情況下,透過DNA外的分子修飾,造成DNA的結構上的改變,進而影響基因活化或表現的修飾作用。然而這類的修飾作用包含了甲基化(methylation)、去甲基化(demethylation)、磷酸化(phosphorylation)、去磷酸化(dephosphorylation)、乙醯化(acetylation)、去乙醯化(deacetylation)、泛素化(ubiquitination)等。這其中甲基化更是常見的修飾作用,因此本研究第一部分即是利用啤酒酵母菌進行甲基轉移酶的探討。

根據Steven Clarke所提出一份經由序列及數學演算分析可能具有甲基轉移酶活性名單,我們挑選了感興趣的啤酒酵母菌基因YNR029C進行深入探討。我們利用了重組蛋白技術及His-tag純化,純化出YNR029Cp並與去除YNR029C基因之啤酒酵母菌(統稱△YNR029C)受質進行甲基化活性分析。結果表示啤酒酵母菌基因YNR029C目前尚無法利用本研究的方法證實其具有蛋白質甲基轉移酶的活性。

本研究第二個部份觀察基因表現與胞器形態之間的關係。我們所著重的是啤酒酵母菌中ADH3p對粒線體形態所造成的影響。我們將建構之ADH3-pYES2、ADH3G211A- pYES2;ADH3-GFP- pYES2、ADH3G211A-GFP- pYES2轉形至酵母菌(BY4742、BJ2168、△YLL001W),並進行蛋白質表現誘導及利用螢光顯微鏡觀察過度表現ADH3p對粒線體形態的影響。結果發現當過度表現ADH3p確實會造成粒線體形態發生改變,形態變成較小且數目較多的顆粒狀粒線體。且我們也誘導了ADH3-GFP- pYES2,將粒線體使用紅螢光染色與ADH3-GFP- pYES2所帶之綠螢光蛋白進行對照分析,試著了解ADH3-GFP蛋白於粒線體中的分佈位置與分布量,結果發現粒線體與ADH3-GFP螢光顯示部分重疊,這說明粒線體形態的改變可能與ADH3p的活性有關。同時我們使用流式細胞儀對ADH3-GFP及ADH3G211A-GFP進行蛋白質定量分析。根據流式細胞儀的結果表示,過度表現ADH3p造成粒線體形態改變是受蛋白質活性影響。
關鍵字:甲基轉移酶、甲基化、粒線體形態、酒精去氫酶III(ADH3)
英文摘要 Epigenetic regulation is causing DNA structural changes by modifier which leaves DNA sequence unchanged. Further epigenetic regulation affects gene activation or expression. Therefore, this modification includes methylation), demethylation, phosphorylation, dephosphorylation, acetylation, deacetylation, and ubiquitination. The methylation is the most common of modification. Consequently, my first part of research is using Saccharomyces cerevisiae to proceed the study of methyltransferase.

According to Steven Clarke who provided a list which shows potential methyltransferase activities by mathematical calculation and analysis of sequences.
We were interested in the Saccharomyces cerevisiae gene YNR029C which was selected for in-depth exploration. We use recombinant protein technology and His-tag purification to purify YNR029Cp. We take the YNR029C-knockout strain Saccharomyces cerevisiae (△YNR029C) as substrate. We take YNR029Cp and the protein extract of △YNR029C for reaction which analyses the methylation activity. The result indicates YNR029Cp does not exhibit protein methyltransferase activity by the methodology employed in this research.

My second part of research is observing relationship of gene expression with organelle morphology. We stress on Saccharomyces cerevisiae ADH3p affecting mitochondrial morphology. We transform yeast strains (BY4742, BJ2168 and △YLL001W) with ADH3-pYES2, ADH3G211A- pYES2, ADH3-GFP- pYES2 and ADH3G211A-GFP- pYES2, and induce protein expression. We use fluorescence microscope to observe mitochondrial morphology upon overexperessing ADH3p. The overexpression of ADH3p indeed effects mitochondrial morphology change. This change is in the size of mitochondria which become smaller and the number increases. Then we also induce ADH3-GFP- pYES2 expression, and use red fluorescent to stain mitochondria superimposed with ADH3-GFPp image, trying to understand the spatial distribution of ADH3-GFP protein relative to the mitochondrial localization. We found that mitochondria and ADH3-GFP fluorescence overlap partially, indicating that mitochondrial morphological changes may be due to ADH3p of activity. We use flow cytometery to analyze the ADH3-GFP and ADH3G211A-GFP protein quantitatively. The results of flow cytometery support that overexpression ADH3p causing changes in mitochondrial morphology is effected by activity of the protein.
論文目次 謝誌………………………………………………………………………I
中文摘要………………………………………………………………III
英文摘要………………………………………………………………V
目錄……………………………………………………………………VII
第一部分 啤酒酵母菌YNR029Cp的表現和純化及甲基化活性之研究
緒論………………………………………………………………………1
材料與方法………………………………………………………………3
1. 材料 ……………………………………………………………3
1.1 菌株…………………………………………………………………3
1.1.1 大腸桿菌品系……………………………………………………3
1.1.2 啤酒酵母菌品系…………………………………………………3
1.2 質體…………………………………………………………………3
1.3 培養基………………………………………………………………4
1.4 實驗儀器……………………………………………………………4
2. 方法 ……………………………………………………………5
2.1 啤酒酵母菌基因YNR029C的製備………………………………5
2.1.1 啤酒酵母菌genomic DNA之萃取………………………………5
2.1.2 YNR029C基因之引子設計……………………………………6
2.1.3 聚合酶鏈鎖反應(polymerase chain reaction, PCR)放大目標基因YNR029C…………………………………………………………………6
2.1.4 使用DNA瓊脂糖膠電泳確認PCR產物…………………………7
2.1.5 純化YNR029C之PCR產物………………………………………8
2.1.6 A-Tailing…………………………………………………………9
2.1.7 TA-cloning 酶接反應(ligation) …………………………………10
2.1.8 轉形至勝任細胞…………………………………………………11
2.1.9 菌種保存…………………………………………………………12
2.1.10.1 質體萃取(傳統法) …………………………………………12
2.1.10.2 質體萃取……………………………………………………13
2.1.11 以限制酶進行確認基因片段…………………………………14
2.1.12 基因序列比對…………………………………………………14
2.1.13 次選殖表現載體pET28c製備…………………………………15
2.1.14 次選殖基因的製備……………………………………………15
2.1.15 次選殖酶接反應………………………………………………16
2.1.16 次選殖質體轉型至DH5α……………………………………16
2.2 重組質體蛋白質表現與純化……………………………………17
2.2.1 各時間點蛋白質表現……………………………………………18
2.2.2 超音波破菌法……………………………………………………18
2.2.3 SDS膠體電泳……………………………………………………19
2.2.4 His-tag 純化重組蛋白質………………………………………19
2.2.5 MALDI-TOF MS分析YNR029Cp………………………………20
2.3 甲基化受質(substrate)的製備……………………………………21
2.3.1 甲基轉移酶甲基化反應…………………………………………22
2.3.2 甲基化反應分析…………………………………………………23
結果與討論……………………………………………………………24
圖與表…………………………………………………………………29
圖(一) YNR029C聚合酶鏈鎖反應(PCR) 電泳圖……………………29
圖(二) A-Tailing後純化電泳圖………………………………………30
圖(三) TA-cloning plasmid及EcoRI切plamsid 電泳圖………………31
圖(四) 台大定序結果…………………………………………………32
圖(五) 限制酶酵素EcoRI、NdeI處理TA-cloning plasmid及pET28c…34
圖(六) 限制酶酵素EcoRI、NdeI確認YNR029C接上pET28c質體……35
圖(七) 含有YNR029C- pET28c plasmid及pET28c plasmid之BL21 DE3菌株誘導8、12小時……………………………………………………36
圖(八) 含有YNR029C- pET28c plasmid及pET28c plasmid之BL21 DE3菌株誘導16、20小時……………………………………………37
圖(九) 含有YNR029C- pET28c plasmid及pET28c plasmid之Rosetta DE3菌株誘導8、12小時………………………………………………38
圖(十) 含有YNR029C- pET28c plasmid及pET28c plasmid之Rosetta DE3菌株誘導16、20小時……………………………………………39
圖(十一) YNR029C- pET28c及pET28c誘導8小時His-tag純化結果…40
圖(十二) MALDI-TOF MS分析YNR029Cp結果…………………41
圖(十三) 甲基化活性測試結果………………………………………43
第二部分 過度表現啤酒酵母菌ADH3p對粒線體形態影響
緒論………………………………………………………………44
1. 材料 ……………………………………………………………46
1.1 菌株………………………………………………………………46
1.1.1 啤酒酵母菌品系…………………………………………………46
1.2 質體………………………………………………………………46
1.3 培養基……………………………………………………………47
1.4 實驗儀器…………………………………………………………47
2. 方法 ……………………………………………………………47
2.1 酵母菌勝任細胞製備……………………………………………47
2.2 質體轉形…………………………………………………………48
2.3 轉形質體蛋白質表現……………………………………………49
2.4 粗抽蛋白質………………………………………………………50
2.5 流式細胞儀測量GFP蛋白螢光強度……………………………50
3. 觀察粒線體變化……………………………………………………51
3.1 粒線體染色………………………………………………………51
3.2 細胞內GFP分布觀察………………………………………………53
結果與討論 ………………………………………………………54
圖與表…………………………………………………………………56
圖(十四) ADH3-pYES2、ADH3G211A- pYES2 、pYES2 (in BJ2168) 粗抽蛋白質結果…………………………………………………………56
圖(十五) 過度表現ADH3p後粒線體的形態,使用MitoTracker Green FM及MitoTracker Red FM染色………………………………………57
圖(十六) 過度表現ADH3p後粒線體的形態,使用MitoTracker Red FM染色…………………………………………………………………58
圖(十七) 過度表現ADH3-GFP後粒線體的形態,使用MitoTracker Red FM染色……………………………………………………………59
圖(十八) 過度表現ADH3-GFP-pYES2、ADH3G211A-GFP- pYES2 、pYES2 (in BJ2168)之Flow cytometer取樣菌大小分佈圖……………60
圖(十九) Flow cytometer偵測綠螢光強度對數圖…………………61
結論……………………………………………………………………62
附錄……………………………………………………………………63
附錄(一) pGEM®-T Easy Vector Map ………………………………63
附錄(二) pET28c Vector Map ………………………………………63
附錄(三) MALDI-TOF MS- Bruker……………………………………64
附錄(四) Flow cytometer偵測綠螢光蛋白定量分析…………………66
附錄(五) pYES2 Vector Map…………………………………………67
參考文獻……………………………………………………………68
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